Treatment Of Paroxysmal Nocturnal Hemoglobinuria Patients By An Inhibitor Of Complement

ABSTRACT

Eculizumab, a humanized monoclonal antibody against C5 that inhibits terminal complement activation, showed activity in a preliminary 12-week open-label trial in a small cohort of patients with paroxysmal nocturnal hemoglobinuria (PNH). The present study examined whether chronic eculizumab therapy could reduce intravascular hemolysis, stabilize hemoglobin levels, reduce transfusion requirements, and improve quality of life in a double-blind, randomized, placebo-controlled, multi-center global Phase III trial. It has been found that eculizumab stabilized hemoglobin levels, decreased the need for transfusions, and improved quality of life in PNH patients via reduced intravascular hemolysis. Chronic eculizumab treatment appears to be a safe and effective therapy for PNH.

BACKGROUND

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologicdisease that results from clonal expansion of hematopoietic stem cellswith somatic mutations in the X-linked gene called PIG-A.^(1,2)Mutations in PIG-A lead to an early block in the synthesis ofglycosylphosphatidylinositol (GPI)-anchors, which are required to tethermany proteins to the cell surface. Consequently, PNH blood cells have apartial (type II) or complete (type III) deficiency of GPI-anchoredproteins.

Intravascular hemolysis is a prominent feature of PNH and a directresult of the absence of the GPI-anchored complement regulatory proteinCD59.^(3,4) Under normal circumstances, CD59 blocks the formation of theterminal complement complex (also called the membrane attack complex) onthe cell surface, thereby preventing erythrocyte lysis and plateletactivation.⁵⁻⁸ Excessive or persistent intravascular hemolysis in PNHpatients not only results in anemia (normal ranges of hemoglobin are14-18 g/dL for men and 12-16 g/dL for women, and persons with lowerlevels are considered to be anemic), but also hemoglobinuria andclinical sequelae related to the release of the erythrocyte contentsinto the circulation: fatigue, thrombosis, abdominal pain, dysphagia,erectile dysfunction, and pulmonary hypertension.^(9,10,21,22) Indeed,impaired quality of life in PNH is disproportionate to the degree ofanemia. Many PNH patients depend on blood transfusions to maintainadequate erythrocyte hemoglobin levels. There have been no therapiesthat effectively reduce intravascular hemolysis and improve theassociated clinical morbidities in PNH.

Eculizumab is a humanized monoclonal antibody directed against theterminal complement protein C5.¹¹ In a preliminary, 12-week, open-labelclinical study in 111 PNH patients, eculizumab was shown to reduceintravascular hemolysis and transfusion requirements.¹² However, thisunblinded study involved a small number of patients with no control armand without protocol-driven transfusion standards.

SUMMARY

The present pivotal, phase III study, Transfusion Reduction Efficacy andSafety Clinical Investigation, Randomized, Multi-Center, Double-Blind,Placebo-Controlled, Using Eculizumab in Paroxysmal NocturnalHemoglobinuria (TRIUMPH), evaluated the effect of eculizumab on thestabilization of hemoglobin levels and transfusion requirements during 6months of treatment in a cohort of 87 transfusion-dependent PNHpatients. Measures of intravascular hemolysis and quality of life werealso assessed. This is the first placebo controlled study of a PNHpatient population to control hemolysis and to differentiate between theeffects due to hemolysis and the effects due to anemia.

It has been surprisingly discovered that certain aspects of quality oflife were unexpectedly improved by the treatment of PNH patients witheculizumab. Furthermore, these improvements in the quality of life wereindependent of transfusion. The improved aspects include, e.g., globalhealth status, physical functioning, emotional functioning, cognitivefunctioning, role functioning, social functioning, fatigue, pain,dyspnea, appetite loss and insomnia. Improvement was also seen in nauseaand vomiting, diarrhea, constipation, and financial difficulties but didnot reach the level of statistical significance. Because the treatedpatients remained anemic throughout their treatment, it was unexpectedthat all of these improvements would have been seen because they werepreviously thought to be a result of the patient being anemic. Althoughnot wishing to be bound by any theory, it appears that some of thesymptoms are likely due, at least in part, to hemolysis and release ofhemoglobin into the bloodstream and do not result solely from thepatient being anemic. The treatment with eculizumab decreases the amountof lysis thereby limiting hemoglobin release into the bloodstream,thereby apparently resulting in the improvements seen in the treatedpatients' quality of life. The results presented herein indicate thatany treatment that decreases hemolysis in a patient will result in animprovement in the quality of life of said patient.

In certain aspects, the application provides a method to improve atleast one aspect of the quality of life of a patient suffering fromparoxysmal nocturnal hemoglobinuria, said method comprisingadministering to said patient in need thereof a compound which inhibitscomplement or inhibits formation of C5b-9.

In certain aspects, the application provides a method to improve atleast one aspect of the quality of life of a patient suffering fromparoxysmal nocturnal hemoglobinuria, said method comprisingadministering to said patient in need thereof a compound which inhibitsintravascular hemolysis. In certain embodiments, said method results ina greater than 30% reduction in LDH in said patient.

In certain aspects, the application provides a method to improve atleast one aspect of the quality of life of an anemic patient whoseanemia results at least in part from hemolysis, said method comprisingadministering to said patient in need thereof a compound which inhibitsintravascular hemolysis, wherein said patient remains anemic. In certainembodiments, said method results in a greater than 30% reduction in LDHin said patient.

In certain aspects, the application provides a method of prolonging thehealth-adjusted life expectancy of a patient comprising administering tosaid patient in need thereof a compound which inhibits formation ofC5b-9. In certain embodiments, said patient is anemic. In certainembodiments, said patient remains anemic following treatment. In certainembodiments, said patient has a hemoglobin level less than i) 14 g/dL ifa man or ii) 12 g/dL if a woman. In certain embodiments, said patienthas a hemoglobin level less than i) 13 g/dL if a man or ii) 11 g/dL if awoman. In certain embodiments, said patient has a hemoglobin level lessthan i) 12 g/dL if a man or ii) 10 g/dL if a woman. In certainembodiments, said patient suffers from paroxysmal nocturnalhemoglobinuria.

In certain aspects, the application provides a pharmaceuticalcomposition comprising an antibody that binds C5 or an active antibodyfragment thereof. In certain embodiments, the antibody that binds C5 oran active antibody fragment thereof is eculizumab. In certainembodiments, the antibody that binds C5 or an active antibody fragmentthereof is pexelizumab. In certain embodiments, the pharmaceuticalformulations of the application may be administered to a subject,particularly a subject having PNH.

In certain aspects, the application provides a method of treating apatient suffering from paroxysmal nocturnal hemoglobinuria byadministering a pharmaceutical composition comprising an antibody thatbinds C5 or an active antibody fragment thereof. In certain embodiments,the antibody that binds C5 or an active antibody fragment thereof iseculizumab. In certain embodiments, the antibody that binds C5 or anactive antibody fragment thereof is pexelizumab. In certain embodiments,the pharmaceutical formulations of the application may be administeredto a subject, particularly a subject having PNH.

In certain aspects, the application provides kits comprising apharmaceutical composition of the application. In some embodiments, thekit further comprises at least one component of a closed sterile system.Components of the closed sterile system include, but are not limited to,needles, syringes, catheter based syringes, needle based injectiondevices, needle-less injection devices, filters, tubing, valves andcannulas. In a related embodiment, the kit comprise components for theremoval of a preservative from the composition. Such components includefilters, syringes, vials, containers, tubing, etc.

In certain embodiments, said quality of life is measured by aFACIT-Fatigue score. In certain embodiments, the FACIT-Fatigue scoreincreases by at least 3 points. In certain embodiments, theFACIT-Fatigue score increases by ≧4 points.

In certain embodiments, said quality of life is measured by an EORTCQLQ-C30 score. In certain embodiments, said EORTC QLQ-C30 score improvesby ≧10% of the pretreatment score. In certain embodiments, said aspectof the quality of life as measured by an EORTC QLQ-C30 score is selectedfrom the group consisting of a) global health status, b) physicalfunctioning, c) emotional functioning, d) cognitive functioning, e) rolefunctioning, f) social functioning, g) fatigue, h) pain, i) dyspnea, j)appetite loss, and k) insomnia. In certain embodiments, said aspect ofquality of life is fatigue.

In certain embodiments, said compound is selected from the groupconsisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venomfactor, FUT-175, complestatin, and K76 COOH. In certain embodiments,said compound is a steroid that suppresses complement.

In certain embodiments, said compound is selected from the groupconsisting of antibodies, active antibody fragments, soluble complementinhibitory compounds, proteins, soluble complement inhibitors with alipid tail, protein fragments, peptides, small organic compounds, RNAaptamers, L-RNA aptamers, spiegelmers, antisense compounds, serineprotease inhibitors, double stranded RNA, small interfering RNA, lockednucleic acid inhibitors, and peptide nucleic acid inhibitors. In certainembodiments, said compound is an antibody or an active antibodyfragment. In certain embodiments, said antibody or active antibodyfragment is selected from the group consisting of a) polyclonalantibodies, b) monoclonal antibodies, c) single chain antibodies, d)chimeric antibodies, e) humanized antibodies, f) Fabs, g) F(ab′)s, h)F(ab′)₂s, i) Fvs, j) diabodies, and k) human antibodies.

In certain embodiments, said antibody or an active antibody fragmentthereof binds C5. In certain embodiments, said antibody or activeantibody fragment blocks C5 cleavage. In certain embodiments, saidantibody or active antibody fragment inhibits the formation of C5b-9. Incertain embodiments, said antibody is eculizumab. In certainembodiments, said antibody or active antibody fragment is administeredfor at least 6 months. In certain embodiments, said patient has aplasticanemia or myelodysplastic syndrome.

In certain embodiments, said antibody that binds C5 or an activeantibody fragment thereof is administered in a single unit dosage form.In certain embodiments, the single unit dosage form is a 300 mg unitdosage form. In certain embodiments, the single unit dosage form islyophilized. In certain embodiments, the single unit dosage form is asterile solution. In certain embodiments, the single unit dosage form isa preservative free formulation. In certain embodiments, the 300 mgsingle-use dosage form comprises 30 ml of a 10 mg/ml sterile,preservative free solution.

In certain embodiments, the antibody that binds C5 or an active antibodyfragment thereof comprises an altered constant region, wherein saidantibody or antigen-binding fragment exhibits decreased effectorfunction relative to an anti-CDCP1 antibody with a native constantregion. In certain embodiments, decreased effector function comprisesone or more properties of the following group: a) decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), and b) decreasedcomplement dependent cytotoxicity (CDC), compared to an anti-CDCP1antibody with a native constant region. In certain embodiments, thealtered constant region comprises a G2/G4 construct in place of the G1domain.

In certain embodiments, the antibody that binds C5 or an active antibodyfragment thereof comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesone or more CDR regions having an amino acid sequence selected from thegroup consisting of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, andwherein the light chain variable region comprises one or more CDRregions having an amino acid sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In certain embodiments,the antibody that binds C5 or an active antibody fragment thereofcomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region consists of SEQ ID NO: 1and the light chain variable region consists of SEQ ID NO: 3. In certainembodiments, the pharmaceutical composition comprises eculizumab. Incertain embodiments, the pharmaceutical composition comprisespexelizumab. In certain embodiments, the antibody that binds C5 or anactive antibody fragment thereof comprises a heavy chain and a lightchain, wherein the heavy chain consists of SEQ ID NO: 2 and the lightchain consists of SEQ TD NO: 4.

In certain embodiments, said patient is anemic. In certain embodiments,said patient remains anemic following treatment. In certain embodiments,said patient has a hemoglobin level less than i) 14 g/dL if a man or ii)12 g/dL if a woman. In certain embodiments, said patient has ahemoglobin level less than i) 13 g/dL if a man or ii) 11 g/dL if awoman. In certain embodiments, said patient has a hemoglobin level lessthan i) 12 g/dL if a man or ii) 10 g/dL if a woman.

In certain embodiments, said health-adjusted life expectancy is measuredaccording to a unit selected from the group consisting of Years ofpotential life lost, Disability-free life expectancy, Health-adjustedlife year, Quality adjusted life year, Healthy years equivalents,Healthy days gained, Episode-free day, Q-TWiST, Health Utilities Index,or Years of healthy life.

In certain embodiments, the health-adjusted life expectancy in a subjectis prolonged by at least one day. In certain embodiments, thehealth-adjusted life expectancy in a subject is prolonged by at leastweek. In certain embodiments, the health-adjusted life expectancy in asubject is prolonged by at least one month. In certain embodiments, thehealth-adjusted life expectancy in a subject is prolonged by at leastone year.

In certain embodiments, the pharmaceutical composition is in a singleunit dosage form. In certain embodiments, the single unit dosage form isa 300 mg unit dosage form. In certain embodiments, the pharmaceuticalcomposition is lyophilized. In certain embodiments, the pharmaceuticalcomposition is a sterile solution. In certain embodiments, thepharmaceutical composition is a preservative free formulation. Incertain embodiments, the pharmaceutical composition comprises a 300 mgsingle-use formulation of 30 ml of a 10 mg/ml sterile, preservative freesolution. In certain embodiments, the pharmaceutical compositioncomprises an antibody that binds C5 or an active antibody fragmentthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B show that eculizumab treatment decreases intravascularhemolysis and increases PNH type III erythrocytes. FIG. 1A shows thedegree of intravascular hemolysis in PNH patients, demonstrated by meanlactate dehydrogenase (LDH) levels. FIG. 1B shows the mean proportion ofPNH type III erythrocytes assessed for placebo- and eculizumab-treatedpatients.

FIG. 2 shows the effect of eculizumab treatment on transfusionrequirements in PNH patients. This is a Kaplan-Meier plot of time tofirst transfusion for eculizumab- and placebo-treated patients frombaseline through week 26. The P value is from the log rank analysis.

FIG. 3 shows the effect of eculizumab on fatigue assessed by theFACIT-Fatigue Instrument. Quality of Life scores were assessed using theFunctional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue)instrument. Values for change from baseline to 26 weeks representleast-square means. A positive change indicates an improvement and anegative change indicates deterioration in the FACIT-Fatigue measures ofquality of life.

DETAILED DESCRIPTION I. Definitions

The term “derived from” means “obtained from” or “produced by” or“descending from”.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques to thisapplication, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

The term “an antigen-binding fragment of an antibody” refers to anyportion of an antibody that retains the binding utility to the antigen.An exemplary antigen-binding fragment of an antibody is the heavy chainand/or light chain CDR, or the heavy and/or light chain variable region.

The term “homologous,” in the context of two nucleic acids orpolypeptides refers to two or more sequences or subsequences that haveat least about 85%, at least 90%, at least 95%, or higher nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using the following sequence comparisonmethod and/or by visual inspection. In certain embodiments, the“homolog” exists over a region of the sequences that is about 50residues in length, at least about 100 residues, at least about 150residues, or over the full length of the two sequences to be compared.

Methods of determining percent identity are known in the art. “Percent(%) sequence identity” with respect to a specified subject sequence, ora specified portion thereof, may be defined as the percentage ofnucleotides or amino acids in the candidate derivative sequenceidentical with the nucleotides or amino acids in the subject sequence(or specified portion thereof), after aligning the sequences andintroducing gaps, if necessary to achieve the maximum percent sequenceidentity, as generated by the program WU-BLAST-2.0a19 (Altschul et al.,J. Mol. Biol. 215:403-410 (1997);http://blast.wustl.edu/blast/README.htm-l) with search parameters set todefault values. The HSP S and HSP S2 parameters are dynamic values andare established by the program itself depending upon the composition ofthe particular sequence and composition of the particular databaseagainst which the sequence of interest is being searched. A “% identityvalue” is determined by the number of matching identical nucleotides oramino acids divided by the sequence length for which the percentidentity is being reported.

II. Overview

The present disclosure relates to a method of treating paroxysmalnocturnal hemoglobinuria (“PNH”), more specifically to improving certainaspects of quality of life which are impaired in PNH patients, and otherhemolytic diseases in mammals. Specifically, the methods of treatinghemolytic diseases, which are described herein, involve using compoundswhich bind to or otherwise block the generation and/or activity of oneor more complement components. The present methods have been found toprovide surprising results. For instance, hemolysis rapidly ceases uponadministration of the compound which binds to or otherwise blocks thegeneration and/or activity of one or more complement components, withhemoglobinuria being significantly reduced after treatment. Also,hemolytic patients can be rendered less dependent on transfusions ortransfusion-independent for extended periods (twelve months or more),well beyond the 120 day life cycle of red blood cells. In addition, typeIII red blood cell count can be increased dramatically in the midst ofother mechanisms of red blood cell lysis (non-complement mediated and/orearlier complement component mediated e.g., Cb3). Another example of asurprising result is that symptoms resolved, indicating that NO serumlevels were increased enough even in the presence of other mechanisms ofred blood cell lysis. These and other results reported herein areunexpected and could not be predicted from prior treatments of hemolyticdiseases.

III The Complement System

The complement system, useful complement inhibitors, and use of theseinhibitors to treat PNH and other patients are more fully described inPCT Patent Application PCT/US2005/003225 filed Feb. 3, 2005 andpublished as International Publication Number WO 2005/074607 A2 on Aug.18, 2005, the contents of which are incorporated herein by reference intheir entirety.

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions.

The complement cascade progresses via the classical pathway or thealternative pathway. These pathways share many components and, whilethey differ in their initial steps, they converge and share the same“terminal complement” components (C5 through C9) responsible for theactivation and destruction of target cells.

The classical complement pathway is typically initiated by antibodyrecognition of and binding to an antigenic site on a target cell. Thealternative pathway is usually antibody independent and can be initiatedby certain molecules on pathogen surfaces. Both pathways converge at thepoint where complement component C3 is cleaved by an active protease(which is different in each pathway) to yield C3a and C3b. Otherpathways activating complement attack can act later in the sequence ofevents leading to various aspects of complement function.

C3a is an anaphylatoxin. C3b binds to bacterial and other cells, as wellas to certain viruses and immune complexes, and tags them for removalfrom the circulation. C3b in this role is known as opsonin. The opsonicfunction of C3b is considered to be the most important anti-infectiveaction of the complement system. Patients with genetic lesions thatblock C3b function are prone to infection by a broad variety ofpathogenic organisms, while patients with lesions later in thecomplement cascade sequence, i.e., patients with lesions that block C5functions, are found to be more prone only to Neisseria infection, andthen only somewhat more prone (Fearon, 1983).

C3b also forms a complex with other components unique to each pathway toform classical or alternative C5 convertase, which cleaves C5 into C5aand C5b. C3 is thus regarded as the central protein in the complementreaction sequence since it is essential to both the alternative andclassical pathways (Wurzner et al., 1991). This property of C3b isregulated by the serun protease Factor 1, which acts on C3b to produceiC3b. While still functional as opsonin, iC3b cannot form an active C5convertase.

C5 is a 190 kDa beta globulin found in normal serum at approximately 75μg/mL (0.4 μM). C5 is glycosylated, with about 1.5-3 percent of its massattributed to carbohydrate. Mature C5 is a heterodimer of a 999 aminoacid 115 kDa alpha chain that is disulfide linked to a 656 amino acid 75kDa beta chain. C5 is synthesized as a single chain precursor proteinproduct of a single copy gene (Haviland et al., 1991). The cDNA sequenceof the transcript of this gene predicts a secreted pro-C5 precursor of1659 amino acids along with an 18 amino acid leader sequence.

The pro-C5 precursor is cleaved after amino acid 655 and 659, to yieldthe beta chain as an amino terminal fragment (amino acid residues +1 to655) and the alpha chain as a carboxyl terminal fragment (amino acidresidues 660 to 1658), with four amino acids deleted between the two.

C5a is cleaved from the alpha chain of C5 by either alternative orclassical C5 convertase as an amino terminal fragment comprising thefirst 74 amino acids of the alpha chain (i.e., amino acid residues660-733). Approximately 20 percent of the II kDa mass of C5a isattributed to carbohydrate. The cleavage site for convertase action isat or immediately adjacent to amino acid residue 733. A compound thatwould bind at or adjacent to this cleavage site would have the potentialto block access of the C5 convertase enzymes to the cleavage site andthereby act as a complement inhibitor.

C5 can also be activated by means other than C5 convertase activity.Limited trypsin digestion (Minta and Man, 1977; Wetsel and Kolb, 1982)and acid treatment (Yamamoto and Gewurz, 1978; Vogt et al., 1989) canalso cleave C5 and produce active C5b.

C5a is another anaphylatoxin. C5b combines with C6, C7, and C8 to formthe C5b-8 complex at the surface of the target cell. Upon binding ofseveral C9 molecules, the membrane attack complex (MAC, C5b-9, terminalcomplement complex-TCC) is formed. When sufficient numbers of MACsinsert into target cell membranes the openings they create (MAC pores)mediate rapid osmotic lysis of the target cells. Lower, non-lyticconcentrations of MACs can produce other effects. In particular,membrane insertion of small numbers of the C5b-9 complexes intoendothelial cells and platelets can cause deleterious cell activation.In some cases activation may precede cell lysis.

As mentioned above, C3a and C5a are anaphylatoxins. These activatedcomplement components can trigger mast cell degranulation, whichreleases histamine and other mediators of inflammation, resulting insmooth muscle contraction, increased vascular permeability, leukocyteactivation, and other inflammatory phenomena including cellularproliferation resulting in hypercellularity. C5a also functions as achemotactic peptide that serves to attract pro-inflammatory granulocytesto the site of complement activation.

The beneficial effect of anti-C5 mAb has previously been reported inseveral experimental models including myocardial reperfusion (Vakeva etal., 1998), systemic lupus erythematosus (Wang et al., 1996) andrheumatoid arthritis (Wang et al., 1995); as well as in human clinicaltrials (Kirschfink, 2001) of autoimmune disease, cardiopulmonary bypassand acute myocardial infarction.

IV Measures of Quality of Life

Various measurements exist to assess quality of life and the effect ofmedical interventions on quality of life for example the Mini-MentalState Examination (MMSE), the Short Test of Mental Status, the EuropeanOrganization for Research and Treatment of Cancer (EORTC) Quality ofLife Questionnaire, the FACIT questionnaires and subscales includingfatigue and anemia, the Likert Scale, and Borg Scale (Tombaugh, et al.,J. Am. Geriatr. Soc. 40:922, 1992; Cummings, JAMA. 269(18):2420, 1993;Crum, et al., JAMA. 269(18):2386, 1993; Folstein, et. al., J. Psychiat.Res. 12:189, 1975; Kokmen, et al., Mayo Clin. Proc. 62:281, 1987;Tang-Wai, et al., Arch. Neurol. 60:1777, 2003; Tamburini, Am. Oncol.12(Suppl. 3):S7, 2001; Webster et al., Health and Quality of LifeOutcomes. 1:79, 2003, www.hqlo.com/content/I/I/79; Grant, et al., Chest.116:1208, 1999; and www.qolid.org). Any of these measurements may beused to assess the change in quality of life due to administration of acompound which inhibits complement or inhibits formation of C5b-9.

In certain embodiments, improvement in quality of life due toadministration of a compound which inhibits complement or inhibitsformation of C5b-9 is measured by the Functional Assessment of ChronicIllness Therapy (FACIT) Measurement System. In certain embodiments,improvement in quality of life is measured by: a) full scales; b)stand-alone subscales; and c) symptom indices.

In certain embodiments, improvement in quality of life due toadministration of a compound which inhibits complement or inhibitsformation of C5b-9 is measured by a European Organization for Researchand Treatment of Cancer (EORTC) Quality of Life Questionnaire. Incertain embodiments, the EORTC questionnaire is the QLQ-C30.

In certain embodiments, improvement in quality of life is measured bythe health-adjusted life expectancy (HALE) index as described inWilkins, R. and Adams, O B., Am J Public Health, 73:1073-1080 (1983).Health-adjusted life expectancy is an average of the quality-adjustedlife years (QALY) for a given population and can be used to evaluate thetherapeutic value of a medical intervention. Quality-adjusted life yearsis a health index that weighs each year of life on a scale from 1 to 0(Weinstein M C and Stason W B, N Engl J Med, 296:716-721 (1977)).Perfect health is rated as 1, death is rated as 0, and disability andpain are rated based on severity. QALY is determined by multiplying thenumber of years at each health status.

In certain embodiments, improvement in quality of life is measured bythe following instruments: Years of potential life lost, Disability-freelife expectancy, Health-adjusted life year, Quality adjusted life year,Healthy years equivalents, Healthy days gained, Episode-free day,Q-TWiST, Health Utilities Index, and Years of healthy life. Thesemeasurements account for both changes in mortality as well as changes inmorbidity and disability. Any of these measurements may be used toassess the change in quality of life due to administration of a compoundwhich inhibits complement or inhibits formation of C5b-9.

In one embodiment, the disclosed methods improve the quality of life ofa patient for at least one day, at least one week, at least two weeks,at least three weeks, at least one month, at least two months, at leastthree months, at least 6 months, at least one year, at least 18 months,at least two years, at least 30 months, or at least three years, or theduration of treatment.

In certain embodiments, the symptoms used to measure quality of life arescaled for intensity. In certain embodiments, the symptoms are scaledfor frequency. In certain embodiments, the symptoms are scaled forintensity and frequency.

In certain aspects, the application provides a method for prolonging thehealth-adjusted life expectancy of a subject comprising administering tothe subject a compound which inhibits complement or inhibits formationof C5b-9. The above measurements account for both changes in mortalityas well as changes in morbidity and disability. Any of thesemeasurements may be used to assess the change in quality-adjusted lifeexpectancy due to administration of a compound which inhibits complementor inhibits formation of C5b-9.

In one embodiment, the disclosed methods prolong the health-adjustedlife expectancy in a subject by at least one day, at least one week, atleast two weeks, at least three weeks, at least one month, at least twomonths, at least three months, at least 6 months, at least one year, atleast 18 months, at least two years, at least 30 months, or at leastthree years as measured by the health-adjusted life expectancy (HALE)index as described in Wilkins et al. Am J Public Health, 73:1073-1080(1983). Health-adjusted life expectancy is an average of thequality-adjusted life years (QALY) for a given population and can beused to evaluate the therapeutic value of a medical intervention.Quality-adjusted life years is a health index that weighs each year oflife on a scale from 1 to 0 (Weinstein et al., N Engl J Med, 296:716-721(1977)). Perfect health is rated as 1, death is rated as 0, anddisability and pain are rated based on severity. QALY is determined bymultiplying the number of years at each health status.

V Inhibitors of the Complement Cascade

In certain embodiments, any compound which binds to or otherwise blocksthe generation and/or activity of one or more complement components canbe used in the present methods. In certain embodiments, a complementinhibitor may be a small molecule (up to 6,000 Da in molecular weight),a nucleic acid or nucleic acid analog, a peptidomimetic, or amacromolecule that is not a nucleic acid, a serine protease inhibitor,or a protein. These agents include, but are not limited to, smallorganic molecules, RNA aptamers including ARC187 (which is commerciallyavailable from Archemix Corp., Cambridge, Mass.), L-RNA aptamers,Spiegelmers, antisense compounds, molecules which may be utilized in RNAinterference (RNAi) such as double stranded RNA including smallinterfering RNA (siRNA), locked nucleic acid (LNA) inhibitors, peptidenucleic acid (PNA) inhibitors.

In certain embodiments, a complement inhibitor may be a protein orprotein fragment. Proteins are known which inhibit the complementcascade, including CD59, CD55, CD46 and other inhibitors of C8 and C9(see, e.g., U.S. Pat. No. 6,100,443). Proteins known as complementreceptors and which bind complement are also known (see, Published PCTPatent Application WO 92/10205 and U.S. Pat. No. 6,057,131). Use ofsoluble forms of complement receptors, e.g., soluble CR1, can inhibitthe consequences of complement activation such as neutrophil oxidativeburst, complement mediated neural injury, and C3a and C5a production. Incertain embodiments, a complement inhibitor may be naturally occurringor soluble forms of complement inhibitory compounds such as CR1,LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175,complestatin, and K76 COOH. Those of skill in the art recognize theabove as some, but not all, of the known methods of inhibitingcomplement and its activation.

In certain embodiments, a complement inhibitor may be an antibodycapable of inhibiting complement, such as an antibody that can block theformation of MAC. For example, an antibody complement inhibitor mayinclude an antibody that binds C5. Such anti-C5 antibodies may directlyinteract with C5 and/or C5b, so as to inhibit the formation of and/orphysiologic function of C5b.

Suitable anti-C5 antibodies are known to those of skill in the art.Antibodies can be made to individual components of activated complement,e.g., antibodies to C7, C9, etc. (see, e.g., U.S. Pat. No. 6,534,058;published U.S. patent application US 2003/0129187; and U.S. Pat. No.5,660,825). U.S. Pat. No. 6,355,245 teaches an antibody which binds toC5 and inhibits cleavage into C5a and C5b thereby decreasing theformation not only of C5a but also the downstream complement components.

The concentration and/or physiologic activity of C5a and C5b in a bodyfluid can be measured by methods well known in the art. For C5a suchmethods include chemotaxis assays, RIAs, or ELISAs (see, for example,Ward and Zvaifler, J Clin Invest. 1971 March; 50(3):606-16; Wurzner, etal., Complement Inflamm. 8:328-340, 1991). For C5b, hemolytic assays orassays for soluble C5b-9 as discussed herein can be used. Other assaysknown in the art can also be used. Using assays of these or othersuitable types, candidate antibodies capable of inhibiting complementsuch as anti-C5 antibodies, now known or subsequently identified, can bescreened in order to 1) identify compounds that are useful in thepractice of the application and 2) determine the appropriate dosagelevels of such compounds.

An antibody capable of inhibiting complement such as an antibody thatbinds C5 affecting C5b is preferably used at concentrations providingsubstantial reduction (i.e., reduction by at least about 25% as comparedto that in the absence of the antibody that binds C5) in the C5b levelspresent in at least one blood-derived fluid of the patient followingactivation of complement within the fluid. Such concentrations can beconveniently determined by measuring the cell-lysing ability (e.g.,hemolytic activity) of complement present in the fluid or the levels ofsoluble C5b-9 present in the fluid. Accordingly, a specificconcentration for an antibody that affects C5b is one that results in asubstantial reduction (i.e., a reduction by at least about 25%) in thecell-lysing ability of the complement present in at least one of thepatient's blood-derived fluids. Reductions of the cell-lysing ability ofcomplement present in the patient's body fluids can be measured bymethods well known in the art such as, for example, by a conventionalhemolytic assay such as the hemolysis assay described by Kabat and Mayer(eds), “Experimental Immunochemistry, 2d Edition”, 135-240, Springfield,Ill., CC Thomas (1961), pages 135-139, or a conventional variation ofthat assay such as the chicken erythrocyte hemolysis method describedbelow.

Specific antibodies capable of inhibiting complement, such as anantibody that binds C5, are relatively specific and do not block thefunctions of early complement components. In particular, such specificagents will not substantially impair the opsonization functionsassociated with complement component C3b, which functions provide ameans for clearance of foreign particles and substances from the body.

C3b is generated by the cleavage of C3, which is carried out byclassical and/or alternative C3 convertases and results in thegeneration of both C3a and C3b. Therefore, in order not to impair theopsonization functions associated with C3b, specific antibodies capableof inhibiting complement such as an antibody that binds C5 do notsubstantially interfere with the cleavage of complement component C3 ina body fluid of the patient (e.g., serum) into C3a and C3b. Suchinterference with the cleavage of C3 can be detected by measuring bodyfluid levels of C3a and/or C3b, which are produced in equimolar ratiosby the actions of the C3 convertases. Such measurements are informativebecause C3a and C3b levels will be reduced (compared to a matched samplewithout the antibody capable of inhibiting complement such as anantibody that binds C5) if cleavage is interfered with by an antibodycapable of inhibiting complement such as an antibody that binds C5.

In practice, the quantitative measurement of such cleavage is generallymore accurate when carried out by the measurement of body fluid C3alevels rather than of body fluid C3b levels, since C3a remains in thefluid phase whereas C3b is rapidly cleared. C3a levels in a body fluidcan be measured by methods well known in the art such as, for example,by using a commercially available C3a EIA kit, e.g., that sold by QuidelCorporation, San Diego, Calif., according to the manufacturer'sspecifications. Particularly specific antibodies capable of inhibitingcomplement such as an antibody that binds C5 produce essentially noreduction in body fluid C3a levels following complement activation whentested in such assays.

Certain antibodies of the disclosure will prevent the cleavage of C5 toform C5a and C5b, thus preventing the generation of the anaphylatoxicactivity associated with C5a and preventing the assembly of the membraneattack complex associated with C5b. As discussed above, in a particularembodiment, these anti-C5 antibodies will not impair the opsonizationfunction associated with the action of C3b.

A preferred method of inhibiting complement activity is to use amonoclonal antibody which binds to complement C5 and inhibits cleavage.This decreases the formation of both C5a and C5b while at the same timeallowing the formation of C3a and C3b which are beneficial to therecipient. Such antibodies which are specific to human complement areknown (U.S. Pat. No. 6,355,245). These antibodies disclosed in U.S. Pat.No. 6,355,245 include a preferred whole antibody (now named eculizumab).A similar antibody against mouse C5 is called BB5.1 (Frei et al., Mol.Cell. Probes. 1:141-149 (1987)). Antibodies to inhibit complementactivity need not be monoclonal antibodies. They can be, e.g.,polyclonal antibodies. They may additionally be antibody fragments. Anantibody fragment includes, but is not limited to, an Fab, F(ab′),F(ab′)₂, single-chain antibody, and Fν. Furthermore, it is well known bythose of skill in the art that antibodies can be humanized (Jones etal., Nature 321:522-S (1986)), chimerized, or deimmunized. Theantibodies to be used in the present disclosure may be any of these. Itis preferable to use humanized antibodies.

In specific embodiments, a therapeutic agent of the disclosure comprisesan antibody or antibody fragment. Antibodies and fragments thereof maybe made by any conventional method, such as those methods describedherein. Antibodies are found in multiple forms, e.g., IgA, IgG, IgM,etc. Additionally, antibodies can be engineered in numerous ways. Theycan be made as single-chain antibodies (including small modularimmunopharmaceuticals or SMIPs™), Fab and F(ab′)₂ fragments, etc.Antibodies can be humanized, chimerized, deimmunized, or fully human.Numerous publications set forth the many types of antibodies and themethods of engineering such antibodies. For example, see U.S. Pat. Nos.6,355,245; 6,180,370; 5,693,762; 6,407,213, 6,548,640; 5,565,332;5,225,539; 6,103,889; and 5,260,203.

This invention provides fragments of anti-C5 antibodies, which maycomprise a portion of an intact antibody, preferably the antigen-bindingor variable region of the intact antibody. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies (Zapata et al., Protein Eng. 8:1057-1062 (1995));single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment of an antibody yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen.

“Fv” refers to the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughlikely at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments that have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: NewYork, 1994), pp. 269-315.

SMIPs are a class of single-chain peptide engineered to include a targetbinding region, effector domain (CH2 and CH3 domains). See, e.g., U.S.Patent Application Publication No. 20050238646. The target bindingregion may be derived from the variable region or CDRs of an antibody,e.g., an antibody that binds C5 of the application. Alternatively, thetarget binding region is derived from a protein that binds C5.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

It is well known that the binding to a molecule (or a pathogen) ofantibodies with an Fc region assists in the processing and clearance ofthe molecule (or pathogen). The Fc portions of antibodies are recognizedby specialized receptors expressed by immune effector cells. The Fcportions of IgG1 and IgG3 antibodies are recognized by Fc receptorspresent on the surface of phagocytic cells such as macrophages andneutrophils, which can thereby bind and engulf the molecules orpathogens coated with antibodies of these isotypes (C. A. Janeway etal., Immunobiology 5th edition, page 147, Garland Publishing (New York,2001)).

This disclosure also provides monoclonal anti-C5 antibodies. Amonoclonal antibody can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey are often synthesized by the hybridoma culture, uncontaminated byother immunoglobulins. Monoclonal antibodies may also be produced intransfected cells, such as CHO cells and NS0 cells. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies and does notrequire production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent disclosure may be made by the hybridoma method first describedby Kohler et al., Nature 256:495-497 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. Nos. 4,816,567 and6,331,415). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597(1991), for example.

A description of the preparation of a mouse anti-human-C5 monoclonalantibody with specific binding characteristics is presented in U.S.Patent Application Publication No. 20050226870. Wurzner et al.,Complement Inflamm. 8:328-340 (1991), describe the preparation of othermouse anti-human-C5 monoclonal antibodies referred to as N19-8 andN20-9.

Other antibodies specifically contemplated are “oligoclonal” antibodies.As used herein, the term “oligoclonal” antibodies” refers to apredetermined mixture of distinct monoclonal antibodies. See, e.g., PCTpublication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In oneembodiment, oligoclonal antibodies consisting of a predetermined mixtureof antibodies against one or more epitopes are generated in a singlecell. In other embodiments, oligoclonal antibodies comprise a pluralityof heavy chains capable of pairing with a common light chain to generateantibodies with multiple specificities (e.g., PCT publication WO04/009618). Oligoclonal antibodies are particularly useful when it isdesired to target multiple epitopes on a single target molecule (e.g.,C5). In view of the assays and epitopes disclosed herein, those skilledin the art can generate or select antibodies or mixtures of antibodiesthat are applicable for an intended purpose and desired need.

In certain embodiments that include a humanized and/or chimericantibody, one or more of the CDRs are derived from an anti-human C5antibody. In a specific embodiment, all of the CDRs are derived from ananti-human C5 antibody. In another specific embodiment, the CDRs frommore than one anti-human C5 antibody are mixed and matched in a chimericantibody. For instance, a chimeric antibody may comprise a CDR1 from thelight chain of a first anti-human C5 antibody combined with CDR2 andCDR3 from the light chain of a second anti-human C5 antibody, and theCDRs from the heavy chain may be derived from a third anti-human C5antibody. Further, the framework regions may be derived from one of thesame anti-human C5 antibodies, from one or more different antibodies,such as a human antibody, or from a humanized antibody. Human orhumanized antibodies are specific for administration to human patients.

In certain embodiments, single chain antibodies, and chimeric, humanizedor primatized (CDR-grafted) antibodies, as well as chimeric orCDR-grafted single chain antibodies, comprising portions derived fromdifferent species, are also encompassed by the present disclosure asantigen-binding fragments of an antibody. The various portions of theseantibodies can be joined together chemically by conventional techniques,or can be prepared as a contiguous protein using genetic engineeringtechniques. For example, nucleic acids encoding a chimeric or humanizedchain can be expressed to produce a contiguous protein. See, e.g., U.S.Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; EuropeanPatent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276 B1;U.S. Pat. No. 5,225,539; and European Patent No. 0,239,400 B1. See also,Newman et al., BioTechnology 10:1455-1460 (1992), regarding primatizedantibody. See, e.g., Ladner et al., U.S. Pat. No. 4,946,778; and Bird etal., Science 242:423-426 (1988), regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, primatized or single chain antibodies, can also beproduced. Functional fragments of the subject antibodies retain at leastone binding function and/or modulation function of the full-lengthantibody from which they are derived. Preferred functional fragmentsretain an antigen-binding function of a corresponding full-lengthantibody (such as for example, ability of antibody that binds C5 to bindC5).

General methods for the immunization of animals (in this case with C5and/or C5b, etc.), isolation of antibody producing cells, fusion of suchcells with immortal cells (e.g., myeloma cells) to generate hybridomassecreting monoclonal antibodies, screening of hybridoma supernatants forreactivity of secreted monoclonal antibodies with a desired antigen (inthis case the immunogen or a molecule containing the immunogen), thepreparation of quantities of such antibodies in hybridoma supernatantsor ascites fluids, and for the purification and storage of suchmonoclonal antibodies, can be found in numerous publications. Theseinclude: Coligan, et al., eds. Current Protocols In Immunology, JohnWiley & Sons, New York, 1992; Harlow and Lane, Antibodies. A LaboratoryManual, Cold Spring Harbor Laboratory, New York, 1988; Liddell andCryer, A Practical Guide To Monoclonal Antibodies, John Wiley & Sons,Chichester, West Sussex, England, 1991; Montz et al., Cellular Immunol.127:337-351 (1990); Wurzner et al., Complement Inflamm. 8:328-340(1991); and Mollnes et al., Scand. J. Immunol. 28:307-312 (1988).

VI Methods of Treatment

Methods of the application may be used to treat paroxysmal nocturnalhemoglobinuria associated symptoms. Methods of the application may beused to treat anemia associated symptoms. Treatment of paroxysmalnocturnal hemoglobinuria and/or anemia may be administered by standardmeans. Treatments of the application may be used in combination withother treatments of the application or known treatments for paroxysmalnocturnal hemoglobinuria and/or anemia. Treatments of the applicationmay be co-administered with other treatments that treat symptoms ofparoxysmal nocturnal hemoglobinuria and/or anemia.

VII Pharmaceutical Formulations and Uses

Methods of administration of small molecules, proteins, and nucleicacids are well-known to those of skill in the art. Methods ofadministration of antibodies are well-known to those of skill in theart. To achieve the desired inhibition, the antibodies can beadministered in a variety of unit dosage forms. The dose will varyaccording to the particular antibody. For example, different antibodiesmay have different masses and/or affinities, and thus require differentdosage levels. Antibodies prepared as Fab fragments will also requirediffering dosages than the equivalent intact immunoglobulins, as theyare of considerably smaller mass than intact immunoglobulins, and thusrequire lower dosages to reach the same molar levels in the patient'sblood. The dose will also vary depending on the manner ofadministration, the particular symptoms of the patient being treated,the overall health, condition, size, and age of the patient, and thejudgment of the prescribing physician. Dosage levels of the antibodiesfor human subjects are generally between about 1 mg per kg and about 100mg per kg per patient per treatment, and preferably between about 5 mgper kg and about 50 mg per kg per patient per treatment. In terms ofplasma concentrations, the antibody concentrations are preferably in therange from about 25 μg/mL to about 500 μg/mL. However, greater amountsmay be required for extreme cases and smaller amounts may be sufficientfor milder cases.

In certain embodiments, the pharmaceutical composition is in a singleunit dosage form. In certain embodiments, the single unit dosage form isa 300 mg unit dosage form. In certain embodiments, the pharmaceuticalcomposition is lyophilized. In certain embodiments, the pharmaceuticalcomposition is a sterile solution. In certain embodiments, thepharmaceutical composition is a preservative free formulation. Incertain embodiments, the pharmaceutical composition comprises a 300 mgsingle-use formulation of 30 ml of a 10 mg/ml sterile, preservative freesolution. In certain embodiments, the antibody is administered accordingto the following protocol: 600 mg via 25 to 45 minute IV infusion every7±2 days for the first 4 weeks, followed by 900 mg for the fifth dose7±2 days later, then 900 mg every 14±2 days thereafter. Antibody isadministered via IV infusion over 25 to 45 minute.

Administration of the anti-C5 antibodies will generally be performed byan intravascular route, e.g., via intravenous infusion by injection.Other routes of administration may be used if desired but an intravenousroute will be the most preferable. Formulations suitable for injectionare found in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 17th ed. (1985). Such formulations must besterile and non-pyrogenic, and generally will include a pharmaceuticallyeffective carrier, such as saline, buffered (e.g., phosphate buffered)saline, Hank's solution, Ringer's solution, dextrose/saline, glucosesolutions, and the like. The formulations may contain pharmaceuticallyacceptable auxiliary substances as required, such as, tonicity adjustingagents, wetting agents, bactericidal agents, preservatives, stabilizers,and the like. In certain embodiments, complement inhibitors such aseculizumab may be administered via IV infusion and diluted to a finalconcentration of 5 mg/ml prior to administration.

Administration of the antibodies capable of inhibiting complement suchas an antibody that binds C5 will generally be performed by a parenteralroute, typically via injection such as intra-articular or intravascularinjection (e.g., intravenous infusion) or intramuscular injection. Otherroutes of administration, e.g., oral (p.o.), may be used if desired andpracticable for the particular antibody capable of inhibiting complementto be administered. Antibodies capable of inhibiting complement such asan antibody that binds C5 can also be administered in a variety of unitdosage forms and their dosages will also vary with the size, potency,and in vivo half-life of the particular antibody capable of inhibitingcomplement being administered. Doses of antibodies capable of inhibitingcomplement such as an antibody that binds C5 will also vary depending onthe manner of administration, the particular symptoms of the patientbeing treated, the overall health, condition, size, and age of thepatient, and the judgment of the prescribing physician.

In certain embodiments, a typical therapeutic treatment includes aseries of doses, which will usually be administered concurrently withthe monitoring of clinical endpoints with the dosage levels adjusted asneeded to achieve the desired clinical outcome. In certain embodiments,treatment is administered in multiple dosages over at least a week. Incertain embodiments, treatment is administered in multiple dosages overat least a month. In certain embodiments, treatment is administered inmultiple dosages over at least a year. In certain embodiments, treatmentis administered in multiple dosages over the remainder of the patient'slife.

The frequency of administration may also be adjusted according tovarious parameters. These include the clinical response, the plasmahalf-life of the therapeutic of the disclosure, and the levels of theantibody in a body fluid, such as, blood, plasma, serum, or synovialfluid. To guide adjustment of the frequency of administration, levels ofthe therapeutic of the disclosure in the body fluid may be monitoredduring the course of treatment.

In certain embodiments, the frequency of administration may be adjustedaccording to an assay measuring cell-lysing ability of complementpresent in one or more of the patient's body fluids. The cell-lysingability can be measured as percent hemolysis in hemolytic assays of thetypes described herein. A 10% or 25% or 50% reduction in the cell-lysingability of complement present in a body fluid after treatment with theantibody capable of inhibiting complement used in the practice of theapplication means that the percent hemolysis after treatment is 90, 75,or 50 percent, respectively, of the percent hemolysis before treatment.

For the treatment of hemolytic diseases such as PNH by systemicadministration of an antibody capable of inhibiting complement such asan antibody that binds C5 (as opposed to local administration),administration of a large initial dose is specific, i.e., a singleinitial dose sufficient to yield a substantial reduction, and morepreferably an at least about 50% reduction, in the hemolytic activity ofthe patient's serum. Such a large initial dose is preferably followed byregularly repeated administration of tapered doses as needed to maintainsubstantial reductions of serum hemolytic titer. In another embodiment,the initial dose is given by both local and systemic routes, followed byrepeated systemic administration of tapered doses as described above.

Formulations particularly useful for antibody-based therapeutic agentsare also described in U.S. Patent App. Publication Nos. 20030202972,20040091490 and 20050158316. In certain embodiments, the liquidformulations of the application are substantially free of surfactantand/or inorganic salts. In another specific embodiment, the liquidformulations have a pH ranging from about 5.0 to about 7.0. In yetanother specific embodiment, the liquid formulations comprise histidineat a concentration ranging from about 1 mM to about 100 mM. In stillanother specific embodiment, the liquid formulations comprise histidineat a concentration ranging from 1 mM to 100 mM. It is also contemplatedthat the liquid formulations may further comprise one or more excipientssuch as a saccharide, an amino acid (e.g., arginine, lysine, andmethionine) and a polyol. Additional descriptions and methods ofpreparing and analyzing liquid formulations can be found, for example,in PCT publications WO 03/106644, WO 04/066957, and WO 04/091658.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the pharmaceuticalcompositions of the application.

In certain embodiments, formulations of the subject antibodies arepyrogen-free formulations which are substantially free of endotoxinsand/or related pyrogenic substances. Endotoxins include toxins that areconfined inside microorganisms and are released when the microorganismsare broken down or die. Pyrogenic substances also includefever-inducing, thermostable substances (glycoproteins) from the outermembrane of bacteria and other microorganisms. Both of these substancescan cause fever, hypotension and shock if administered to humans. Due tothe potential harmful effects, it is advantageous to remove even lowamounts of endotoxins from intravenously administered pharmaceuticaldrug solutions. The Food & Drug Administration (“FDA”) has set an upperlimit of 5 endotoxin units (EU) per dose per kilogram body weight in asingle one hour period for intravenous drug applications (The UnitedStates Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)).When therapeutic proteins are administered in amounts of several hundredor thousand milligrams per kilogram body weight, as can be the case withmonoclonal antibodies, it is advantageous to remove even trace amountsof endotoxin.

Formulations of the subject antibodies include those suitable for oral,dietary, topical, parenteral (e.g., intravenous, intraarterial,intramuscular, subcutaneous injection), ophthalmologic (e.g., topical orintraocular), inhalation (e.g., intrabronchial, intranasal or oralinhalation, intranasal drops), rectal, and/or intravaginaladministration. Other suitable methods of administration can alsoinclude rechargeable or biodegradable devices and controlled releasepolymeric devices. Stents, in particular, may be coated with acontrolled release polymer mixed with an agent of the application. Thepharmaceutical compositions of this disclosure can also be administeredas part of a combinatorial therapy with other agents (either in the sameformulation or in a separate formulation).

The amount of the formulation which will be therapeutically effectivecan be determined by standard clinical techniques. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems. The dosage of the compositions to beadministered can be determined by the skilled artisan without undueexperimentation in conjunction with standard dose-response studies.Relevant circumstances to be considered in making those determinationsinclude the condition or conditions to be treated, the choice ofcomposition to be administered, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms. Forexample, the actual patient body weight may be used to calculate thedose of the formulations in milliliters (mL) to be administered. Theremay be no downward adjustment to “ideal” weight. In such a situation, anappropriate dose may be calculated by the following formula: Dose(mL)=[patient weight (kg)×dose level (mg/kg)/drug concentration (mg/mL)]

To achieve the desired treatment results, anti-C5 antibodies can beadministered in a variety of unit dosage forms. The dose will varyaccording to the particular antibody. For example, different antibodiesmay have different masses and/or affinities, and thus require differentdosage levels. Antibodies prepared as Fab′ fragments or single chainantibodies will also require differing dosages than the equivalentnative immunoglobulins, as they are of considerably smaller mass thannative immunoglobulins, and thus require lower dosages to reach the samemolar levels in the patient's blood.

Other therapeutics of the disclosure can also be administered in avariety of unit dosage forms and their dosages will also vary with thesize, potency, and in vivo half-life of the particular therapeutic beingadministered.

Doses of therapeutics of the disclosure will also vary depending on themanner of administration, the particular symptoms of the patient beingtreated, the overall health, condition, size, and age of the patient,and the judgment of the prescribing physician.

The formulations of the application can be distributed as articles ofmanufacture comprising packaging material and a pharmaceutical agentwhich comprises the antibody capable of inhibiting complement and apharmaceutically acceptable carrier as appropriate to the mode ofadministration. The packaging material may include a label whichindicates that the formulation is for use in the treatment of hemolyticdiseases such as PNH. Although antibodies are preferred, especiallyanti-C5 antibodies which have already been shown to be safe andeffective at decreasing the accumulation of downstream complementcomponents in persons, the use of other complement inhibitors is alsocontemplated by this disclosure. The pharmaceutical formulations anduses of the disclosure may be combined with any known complementinhibitors or hemolytic diseases treatments known in the art.

In certain aspects, the application provides kits comprising apharmaceutical composition of the application. In some embodiments, thekit further comprises at least one component of a closed sterile system.Components of the closed sterile system include, but are not limited to,needles, syringes, catheter based syringes, needle based injectiondevices, needle-less injection devices, filters, tubing, valves andcannulas. In a related embodiment, the kit comprise components for theremoval of a preservative from the composition. Such components includefilters, syringes, vials, containers, tubing, etc.

Exemplification Methods Patient Selection

The TRIUMPH trial consisted of a 2-week screening period, an observationperiod of up to 3 months duration, and a 2-week treatment period.

During the screening period, patients were evaluated with respect toinclusion and exclusion criteria. Men and women, 18 years or older,diagnosed as having PNH with a type III erythrocyte population of ≧10%,and who had received at least 4 transfusions in the previous 12 monthswere eligible. Concomitant administration of erythropoietin,immunosuppressants, corticosteroids, coumadin, low molecular weightheparin, iron supplements, and folic acid were not reasons forexclusion, provided the doses were steady prior to the first visit andthroughout the duration of the study. Because of the increased frequencyof neisserial infections in individuals genetically deficient interminal complement proteins, all patients were vaccinated againstNeisseria meningitides. Patients were to avoid conception. The protocolwas approved by an Investigational Review Board at each clinical siteand written informed consent was obtained from all patients enrolled.Patients transfused with a mean pre-transfusion hemoglobin level >10.5g/dL over the previous 12 months, and those who showed evidence ofhaving a suppressed immune response, complement deficiency, or activebacterial infection, including any history of meningococcal disease,were excluded from the study. Patients were also not eligible if theyhad previously received a bone marrow transplant or if they hadparticipated in another trial or received another investigational drugwithin 30 days of the first visit. An individualized transfusionalgorithm was calculated for each patient based on their prior 12-monthtransfusion history; the written algorithm documented the number ofpacked red blood cell (PRBC) units transfused for given hemoglobinvalues and served as a prospectively determined guide for transfusionduring observation and treatment periods.

Each patient considered eligible entered an observation period of up to13 weeks in order to confirm their PBRC transfusion dependence. At leastone transfusion—termed the “qualifying” transfusion—during the 13-weekobservation period at a hemoglobin value at or below 9 g/dL withsymptoms, or at or below 7 g/dL with or without symptoms, in accordancewith the transfusion algorithm indicated for each patient, was arequirement to proceed to randomization. The hemoglobin value at whicheach individual's qualifying transfusion was administered, was definedas the hemoglobin “set point” for that individual for the purpose of theprimary efficacy variable. A platelet count ≧100,000/mL and a LDH level≧1.5 times the upper limit of the normal range were also required eitherat screening or during the observation period for eligibility.

Study Design

Patients were randomly assigned on a one-on-one basis to receive eitherplacebo or eculizumab (Soliris™, Alexion Pharmaceuticals, Inc.) within10 days of the qualifying transfusion. Study medication was dosed in ablinded fashion as follows: 600 mg eculizumab for patients randomlyassigned to active drug, or placebo for those patients randomly assignedto placebo, respectively via IV infusion every 7±1 days for 4 doses;followed by 900 mg eculizumab, or placebo, respectively, via IV infusion7±1 day later; followed by a maintenance dose of 900 mg eculizumab, orplacebo, respectively, via IV infusion every 14±2 days for a total of 26weeks of treatment.

Measures of Clinical Efficacy

There were two co-primary endpoints in the study: (1) stabilization ofhemoglobin levels, defined as a hemoglobin value maintained above theindividual hemoglobin set point in the absence of transfusions for theentire 26-week treatment period, and (2) reduction in units of PRBCstransfused during the 26-week treatment phase of the study. The triggerfor transfusion during the study period remained unchanged for eachpatient, as compared with their care before entry into the study:patients received blood transfusions when they had symptoms resultingfrom anemia and reached their individualized, pre-determined “setpoint”. Pre-specified secondary endpoints included transfusionavoidance, hemolysis as measured by LDH area under the curve frombaseline to 26 weeks, and QoL changes as measured from baseline to 26weeks using the Functional Assessment of Chronic Illness Therapy-Fatigue(FACIT-Fatigue) instrument.¹³ Pre-specified exploratory analysesincluded assessment of the EORTC QLQ-C30 instrument,¹⁴ the change in LDHfrom baseline through week 26, and thrombosis. Other pre-specifiedmeasurements included pharmacokinetics, pharmacodynamics, andimmunogenicity of eculizumab. Time to first transfusion during the26-week treatment phase and the proportion of PNH type III blood cellswere also assessed.

Safety Assessments

Treatment-emergent adverse events, clinical laboratory tests (e.g.,serum chemical analyses and complete blood counts), electrocardiogramdata, and vital signs were assessed. Adverse events were defined usingthe MedDRA preferred terms and tabulated as incidence rates pertreatment group.

Statistical Analysis

For co-primary endpoints, analyses were performed according to theintention to treat using the data from all patients who were randomizedand received study drug; stabilization of hemoglobin levels was analyzedusing the Fisher's exact test and total PRBC units transfused wereanalyzed with the Wilcoxon's rank sum test. For comparison of treatmenteffect on transfusion avoidance, the Fisher's exact test was used on theincidence and the log rank test was used for time to first transfusion.For LDH area under the curve the Wilcoxon's rank sum test was used.

Quality of life measure of fatigue was assessed using the scoringguidelines for the FACIT-Fatigue instrument.¹⁵ Assessment of quality oflife measures based on the EORTC QLQ-C30 instrument was conducted inaccordance with the appropriate scoring guidelines.¹⁶ The changes ofFACIT-Fatigue and EORTC QLQ-C30 scores from baseline through 26 weekswere analyzed using a mixed model, with baseline as covariate, treatmentand time as fixed effects, and patient as a random effect. Changes inLDH levels and PNH type III erythrocytes from baseline through 26 weekswere analyzed using the same mixed model. Two-sided tests were used forall analyses. The adverse events and long-term safety checklist weretabulated separately and compared between treatments using the Fisher'sexact test. A p-value ≦0.05 was considered to be statisticallysignificant.

Results Characteristics of Patients

A total of 115 PNH patients were screened. Six patients did not meet theinclusion/exclusion criteria during the screening period. Twenty-oneother patients did not receive a qualifying transfusion and were notrandomized into the treatment phase. One patient who did not meet theinclusion criteria was inadvertently randomized, but did not receivestudy medication. Thus 87 hemolytic PNH patients (35 men and 52 women)were enrolled and randomized to receive either eculizumab (N=43) orplacebo (N=44), exceeding the original target of 75 randomized patients.

Patient characteristics were similar in the eculizumab- andplacebo-treated cohorts: median age, 41 (range 20-85) and 35 (range18-78) years; median duration of PNH, 4.2 (range 0.8 to 29.7) and 9.2(range 0.4 to 38.3) years; patients with history of aplastic anemia, 4and 11; history of myelodysplastic syndrome, 1 and 0; and history ofthrombosis, 9 (16 events) and 8 (11 events). Stable usage of concomitantmedications at baseline in the eculizuumab- and placebo-treated groupsincluded the following: erythropoietin, 3 patients and 0 patients;cyclosporine, 1 and 1; anticoagulants (coumarins or heparins) 21 and 11;and steroids (glucocorticoids or androgenic steroids), 12 and 12,respectively.

Of the 87 patients randomized, 85 completed the trial. Two patients whodid not complete the trial had been randomized to the eculizumab arm:one patient discontinued due to the inconvenience of travel to the studysite and the second patient became pregnant. Ten patients in theplacebo-treatment group discontinued infusions, in all cases due toperceived lack of efficacy, but they remained in the study formonitoring purposes.

Pharmacokinetics/Pharmacodynamics

In 42 of the 43 eculizumab-treated patients the levels of drug duringthe maintenance period (900 mg every 2 week±2 days) were sufficient tocompletely block serum hemolytic activity (mean trough value at week 26of 101.8 μg/mL). A single patient did not sustain therapeutic troughlevels of eculizumab and demonstrated a breakthrough in complementblockade during the last few days of each dosing interval. Thesebreakthroughs were clinically manageable and quickly resolved followingthe next dose.

Hemolytic Efficacy Variables

The impact of terminal complement inhibition with eculizumab on chronicintravascular hemolysis in PNH patients was demonstrated in this studyby an immediate (one week) and sustained decrease in mean levels of LDH(FIG. 1A). The median LDH area under the curve during the 26-week studyperiod was reduced 85.8% in eculizumab-relative to placebo-treatedpatients (P<0.001). The mean LDH level decreased from 2199.7±157.7 IU/Lat baseline to 327.3±67.6 IU/L by 26 weeks in eculizumab-treatedpatients while levels in placebo-treated patients remained consistentlyelevated with values of 2259.0±158.5 IU/L at baseline and 2418.9±140.3IU/L at 26 weeks (P<0.001, for eculizumab versus placebo). A secondbiochemical measure of hemolysis, serum aspartate aminotransferase(AST), also showed a statistically significant improvement followingeculizumab- versus placebo-treatment (data not shown). Haptoglobinlevels were statistically significantly increased in eculizumab- ascompared to placebo-treated patients but mean levels of haptoglobin werestill below normal levels in eculizumab-treated patients (data notshown).

FIG. 1A shows the degree of intravascular hemolysis in PNH patients,demonstrated by mean lactate dehydrogenase (LDH) levels (±standarderror) from baseline (study week 0) to week 26 for both eculizumab- andplacebo-treated patient populations. Screening occurred up to 3 monthsprior to study week 0. The upper limit of the normal range (103-223IU/L) for LDH is indicated by a dashed line. LDH was reduced to a meanlevel just above the upper limit of normal at week 26 foreculizumab-treated patients; 15 of 41 patients who completed the studydemonstrated LDH levels within the normal range. All placebo-treatedpatients remained at least 5 times above the upper limit of normal atweek 26. The P value is based on a mixed model analysis from baselinethrough week 26. FIG. 1B shows the mean proportion (±standard error) ofPNH type III erythrocytes assessed for placebo- and eculizumab-treatedpatients. The screening visit occurred up to 3 months prior to studyweek 0. The P value is based on a mixed model analysis from baselinethrough week 26.

A corollary to the reduction in intravascular hemolysis duringeculizumab treatment was an observed increase in the PNH type IIIerythrocyte population (FIG. 1B). The mean proportions of type IIIerythrocytes increased from 28.1±2.0% at baseline to 56.9±3.6% by week26 for eculizumab-treated patients while proportions in the placebogroup remained constant with mean values of 35.7±2.8% before treatmentto 35.5±2.8% at 26 weeks (P<0.001, for eculizumab versus placebo). Bycontrast, the proportions of PNH type III granulocytes and monocytes didnot change significantly between the treatment groups during thetreatment period and were greater than 90% at week 26.

Clinical Efficacy Co-Primary Endpoints

The co-primary efficacy endpoints in the TRIUMPH trial werestabilization of hemoglobin levels and reduction in PRBC unitstransfused. At the end of the treatment period, 48.8% ofeculizumab-treated patients had maintained levels of hemoglobin abovethe pre-specified set-point (median set-point value of 7.7 g/dL for bothtreatment groups) in the absence of transfusions, whereas stabilizationof hemoglobin did not occur in any of the patients in the placebo group(P<0.001; Table 1). By week 26, the median of PRBC units transfused perpatient was 0 in the eculizumab group and 10.0 in the placebo cohort(P<0.001), while the mean of PRBC units transfused was 3.0 and 11.0 inthe eculizumab and placebo cohorts, respectively. In the 6-month periodprior to the study, the median of PRBC units transfused per patient was9.0 in the eculizumab cohort and 8.5 in placebo patients while the meanof PRBC units transfused was 9.6±0.6 and 9.7±0.7, respectively. Meanhemoglobin levels were similar between the treatment groups at baseline(10.0±1.8 g/dL in eculizumab-treated patients and 9.7±1.8 g/dL inplacebo-treated patients) and did not substantially change by week 26(10.1±2.5 g/dL and 8.9±2.2 g/dL in eculizumab and placebo cohorts,respectively).

The median time to first transfusion was not reached during the studyperiod in eculizumab-treated patients (it was greater than 26 weeks)while the placebo group reached the median time to first transfusion inonly 4 weeks (P<0.001; FIG. 2). Transfusion avoidance was achieved in51.2% and 0% of the eculizumab and placebo cohorts, respectively(P<0.001). By the end of the 26-week treatment period, the total PRBCunits transfused were 131 in eculizumab-treated patients versus 482 inthe placebo group (Table 1). By contrast, in the 6-month period prior tothe study, total PRBC units transfused in the eculizumab- andplacebo-cohorts were 413 and 417, respectively.

TABLE 1 Stabilization of Hemoglobin Levels and Reduction in TransfusionRequirements during Eculizumab Treatment PRE-TREATMENT HISTORY*TREATMENT PERIOD Placebo Eculizumab Placebo Eculizumab P Value†Stabilization of Hemoglobin NA NA 0 48.8 <0.001‡ Levels in the absenceof transfusions (percent of patients) †Units Transfused per PatientMedian 8.5 9.0 10 0 <0.001§ Mean ± SE 9.7 ± 0.7 9.6 ± 0.6 11.0 ± 0.833.0 ± 0.67 Total Units Transfused per 417 413 482 131 Group *Twelvemonth historical transfusion data normalized to 6 months †Co-primaryendpoints ‡Base on 2-sided Fisher's exact test §Based on Wilcoxon's ranksum test NA, not applicable

Improvements in Quality of Life Measures

Assessments of quality of life in PNH patients during eculizumabtreatment were performed using two different instruments; FACIT-Fatigueand the EORTC QLQ-C30. Eculizumab-treated patients showed a meanincrease (improvement) in the FACIT-Fatigue score of 6.4±1.2 points frombaseline to 26-weeks while the mean score in placebo patients decreasedby 4.0±1.7 points, a total difference between the treatment groups of10.4 points (FIG. 3). Mixed model analysis of covariance demonstrated astatistically significant difference between treatment groups (P<0.001).

For the EORTC instrument, improvements were observed witheculizumab-treatment in each subscale. Statistically significantimprovements with eculizumab-compared with placebo-treated groups wereobserved in the following quality of life subscales (Table 2): globalhealth status (P<0.001), physical functioning (P<0.001), emotionalfunctioning (P=0.008), cognitive functioning (P=0.002), role functioning(P<0.001), social functioning (P=0.003), fatigue (P<0.001), pain(P=0.002), dyspnea (P<0.001), appetite loss (P<0.001), and insomnia(P=0.014). The improvements with eculizumab treatment in the otherscales, including nausea and vomiting, diarrhea, constipation, andfinancial difficulties, did not reach statistical significance.

TABLE 2 Effect of Eculizumab Treatment on Quality of Life Assessed bythe EORTC QLQ-C30 Instrument Mean Change from Baseline to Week 26*Absolute P Placebo Eculizumab Difference Value† Global Health −8.5 10.919.4 <0.001 Status Functional Role −6.9 17.9 24.8 <0.001 Social 2.0 16.714.6 =0.003 Cognitive −6.1 7.9 14.0 =0.002 Physical −3.5 9.4 13.0 <0.001Emotional −3.7 7.5 11.2 =0.008 Symptoms/Single Items Fatigue 10.0 −16.927.0 <0.001 Pain 5.3 −12.3 17.6 =0.002 Dyspnea 8.9 −7.9 16.9 <0.001Appetite Loss 3.3 −10.3 13.6 <0.001 Insomnia 4.9 −7.9 12.8 =0.014Financial 0.0 −10.3 10.3 =0.186 Difficulties Constipation 0.0 −6.3 6.3=0.199 Nausea/vomiting 2.8 −0.4 3.2 =0.056 Diarrhea 5.7 4.8 0.9 =0.147*A positive change indicates an improvement in global health status andfunctional scales and a negative change indicates an improvement insymptom and single item scales. †Based on a mixed analysis-of-covariancemodel with visit as a fixed effect, patient as a random effect andbaseline as a covariate.

Relationship Between FACIT-Fatigue Quality of Life and IntravascularHemolysis

In order to determine if there was a treatment independent relationshipbetween the FACIT-Fatigue quality of life instrument and intravascularhemolysis, an analysis was performed whereby the mean LDH level (throughthe 26 week study period) for each TRIUMPH patient was analyzed as afunction of the patient's respective mean change in FACIT-Fatigue scorefrom baseline (through the 26 week study period) (see Table 3). For thisanalysis, mean levels of LDH were divided into 4 groups that included:normal levels, 1-2 times the upper limit of normal (ULN), 2-10 times theupper limit of normal, and greater than 10 times the upper limit ofnormal. The analysis demonstrated that patients who maintained normalLDH levels throughout the study experienced significant improvements infatigue when compared to patients who had incrementally higher levels ofLDH throughout the study (p=0.0048). These data establish a clear linkbetween increasing intravascular hemolysis as measured by LDH levels anddecreased quality of life as measured by the FACIT-Fatigue quality oflife instrument.

TABLE 3 Relationship between FACIT-Fatigue and Intravascular HemolysisChange of FACIT Treatment LDH from Baseline Group Category <4 >=4 PValue Combined Normal  4 (30.77%) 9 (69.23%) .0048 1-2 × ULN 16 (61.54%)10 (38.46%)  2-10 × ULN 18 (72.00%) 7 (28.00%) >10 × ULN 16 (80.00%) 4(20.00%)

Safety

There were no deaths in the study. Serious adverse events (SAEs) werereported for 13 patients, of which 4 occurred in the eculizumab-treatedcohort and 9 were in the placebo-treated cohort (see Table 4). Allpatients recovered without sequelae.

The most common AEs reported for eculizumab-treated patients wereheadache, nasopharyngitis, back pain, and upper respiratory tractinfection. Headache and back pain occurred more commonly in theeculizumab-treatment group compared with the placebo group. However, theincrease in headaches was limited to the first 2 weeks of therapy andwas mild to moderate. There were no statistically significantdifferences in incidents rates between treatment groups for any AEsreported.

One episode of thrombosis (Budd-Chiari) occurred in a placebo-treatedpatient. There were no thromboses in eculizumab-treated patients.

Only one patient showed a detectable level of anti-eculizumab antibodiesin the eculizumab-treated cohort; this response was weak (did nottitrate), occurred at only one time point and did not result in adisruption of complement blockade.

TABLE 4 Adverse Event Reporting Placebo Eculizumab Patients Patients n(percent) n (percent) SERIOUS ADVERSE EVENTS* Total 9 (20.5) 4 (9.3)Eculizumab treatment emergent Exacerbation of PNH 3 (6.8) 1 (2.3) Renalcolic 0 1 (2.3) Lumbar sacral disc prolapse 0 1 (2.3) Alphastreptococcal bacteremia 0 1 (2.3) Central line infection and UTI 1(2.3) 0 (0) Upper respiratory tract infection 1 (2.3) 0 (0) Probableviral infection 1 (2.3) 0 (0) Neutropenia 1 (2.3) 0 (0)Cellulitis/folliculitis/ 1 (2.3) 0 (0) neutropenia Anemia and pyrexia 1(2.3) 0 (0) MOST FREQUENT ADVERSE EVENTS*† Headache§ 12 (27.3) 19‡(44.2) Nasopharyngitis 8 (18.2) 10 (23.3) Upper respiratory tractinfection 10 (22.7) 6 (14) Back pain 4 (9.1) 8 (18.6) Nausea 5 (11.4) 7(16.3) Cough 4 (9.1) 5 (11.6) Diarrhea 5 (11.4) 4 (9.3) Arthralgia 5(11.4) 3 (7.0) Abdominal pain 5 (11.4) 2 (4.7) Dizziness 5 (11.4) 2(4.7) Vomiting 5 (11.4) 2 (4.7) Fatigue 1 (2.3) 5 (11.6) Viral infection5 (11.4) 1 (2.3) *By preferred terms †Occurring in 10% or more ofpatients ‡Sixteen of 19 patients experienced headache within 48 hours ofinfusion §Following the first 2 weeks of dosing, 20.9% of eculizumab-and 22.7% of placebo-treated patients experienced headache

DISCUSSION

Chronic intravascular hemolysis with periods of acute exacerbation arethe classical manifestations of PNH, frequently resulting in anemia, theneed for transfusions to sustain hemoglobin levels, and deterioration inquality of life. In the phase III pivotal TRIUMPH study, we examined theeffect of terminal complement inhibition with eculizumab on hemoglobinlevels and transfusion requirements in patients with PNH. Forty-ninepercent of patients treated with eculizumab over the 6-month perioddemonstrated stabilization of hemoglobin in the absence of transfusionscompared to no patients in the placebo arm of the trial. Over 50% ofeculizumab-treated patients were transfusion independent during theentire study compared to none in the placebo arm, and the overall meantransfusion rate was reduced by 73%. Moreover, even in patients who didnot achieve transfusion independence, eculizumab treatment was %associated with a 44% reduction in the rate of transfusion (data nowshown).

Lactate dehydrogenase, a biochemical marker of hemolysis in PNH,⁹ wasimmediately and consistently decreased in all eculizumab-treatedpatients, while patients in the placebo cohort continued to hemolyzewith levels of LDH exceeding 5 times the upper limit of the normal rangein all patients at the study end. Levels of LDH were reduced into thenormal range in approximately one-third of eculizumab-treated patients,while the remainder stabilized at a level just above the upper limit ofnormal suggesting residual low level hemolysis in some patients. Levelsof haptoglobin, a more sensitive marker of the presence of cell freehemoglobin in the circulation, were undetectable in most patients. Lowlevel hemolysis in a subset of eculizumab-treated patients is possiblydue to an inherent decrease in survival of these cells or C3b-mediated,extravascular clearance of PNH erythrocytes through thereticuloendothelial system.¹⁷

Before eculizumab treatment, hemoglobin levels in study patients wereartificially maintained by frequent transfusion. Therefore,stabilization of hemoglobin levels with a concomitant cessation of orreduction in transfusions represents a net increase in endogenoushemoglobin levels. Our data suggest that resolution of hemolysis witheculizumab results in a new steady state hemoglobin level determined bya balance between the extent of the underlying bone marrow dysfunction,the number of PNH erythrocytes that are preserved by eculizumab therapyand the new level (if any) of transfusion requirement.

Patients with PNH generally experience markedly impaired quality of lifecharacterized by fatigue, anemia, thrombosis, and pulmonary hypertensionas well as smooth muscle dystonia including abdominal pain, dysphagia,and erectile dysfunction.^(9,10,18) These symptoms have been attributedto both excessive intravascular hemolysis and downstream scavenging ofnitric oxide by cell free hemoglobin in plasma. The reduction ofintravascular hemolysis in eculizumab-treated patients in the currentstudy was associated with significant improvements in the fatiguecomponent of quality of life relative to placebo-treated patients asassessed via the FACIT-Fatigue instrument. Further, eculizumab therapywas associated with a median increase of 6.4 points over baseline valuesestablished before treatment. It has previously been demonstrated thatan increase of 3 or more points from baseline represents a clinicallyimportant difference in this quality of life instrument.¹⁹ Patients whoreceived eculizumab also experienced a significant improvement in mostdomains of the EORTC QLQ-30 relative to the placebo-treated cohortincluding global health status, physical functioning, emotionalfunctioning, cognitive functioning, role functioning, socialfunctioning, fatigue, pain, dyspnea, appetite loss, and insomnia.Improvement in the fatigue component of the EORTC QLQ-30 providessupport for the improvement demonstrated in the FACIT-Fatigue instrumentduring eculizumab therapy. Importantly, these improvements in quality oflife in the eculizumab-treated patients occurred despite similar levelsof erythrocyte hemoglobin in the two treatment groups, furthersupporting the contribution of hemolysis per se, as opposed to anemia,in mediating the poor quality of life in PNH patients. Clinicalassessment of additional life quality-related symptoms of PNH, includingabdominal pain, dysphagia, and erectile dysfunction, have also beenreported to improve during eculizumab therapy.²⁰

Eculizumab was safe and well-tolerated. There were no deaths in thestudy and only a single thrombotic event which occurred in a placebopatient in a site (the hepatic veins) which is typical of the thrombosisin PNH. The relative brief duration of this study was not sufficient toaddress the relevant issue of a possible protection from thrombosis byterminal complement inhibition with eculizumab.

Adverse events were generally mild with headache occurring at increasedfrequency in the eculizumab-treated patients; however, this increasedfrequency did not persist following the first two doses of therapy.There were 4 SAEs in the eculizumab treatment group and 9 SAEs in theplacebo group. There was no evidence of increased infection risk ineculizumab-treated patients during the study period. Oneeculizumab-treated patient showed a low level of anti-eculizumabantibodies at one time point during the study which did not persist anddid not result in a disruption of complement blockade. There were no AEsassociated with eculizumab withdrawal in the 2 eculizumab-treatedpatients who did not complete the trial. Additional safety assessments,as well as efficacy measures, are being examined in an ongoingmulti-center, open-label Phase III safety trial of eculizumab (SHEPHERD)in approximately 95 patients with PNH.

Results from the current randomized, double-blind, placebo-controlled,global study show that terminal complement inhibition with eculizumabappears to be a safe and effective therapy for patients with the raredisorder PNH. Treatment with eculizumab reduced intravascular hemolysis,and stabilized hemoglobin levels despite a reduction of transfusions, tothe point where most PNH patients were rendered transfusion independent.Substantial and clinically meaningful improvements in fatigue and otherkey quality of life parameters were also demonstrated. All of the 85patients who completed the study elected to receive eculizumab in anopen-label extension study and all currently remain on drug. The resultsof the TRIUMPH study indicate that terminal complement inhibition witheculizumab safely and effectively addresses an important consequence ofthe underlying genetic defect in PNH hematopoietic stem cells byproviding a therapeutic replacement for the terminal complementinhibitor deficiency.

The present invention provides among other things treatment with aninhibitor of complement. Many variations of the invention will becomeapparent to those skilled in the art upon review of this specification.The full scope of the invention should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

AU publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

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SEQUENCES SEQ ID NO: 1-Eculizumab V_(H)QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSA SEQ ID NO: 2-Eculizumab Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 3-EculizumabV_(L) MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRT SEQ ID NO: 4-Eculizumab Light chainMDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5-Eculizumab CDRH1 NYWIQSEQ ID NO: 6-Eculizumab CDRH2 EILPGSGSTEYTENFKD SEQ ID NO: 7-EculizumabCDRH3 YFFGSSPNWYFDV SEQ ID NO: 8-Eculizumab CDRL1 GASENIYGALN SEQ ID NO:9-Eculizumab CDRL2 GATNLAD SEQ ID NO: 10-Eculizumab CDRL3 QNVLNTPLT

1. A method to improve at least one aspect of the quality of life of apatient suffering from paroxysmal nocturnal hemoglobinuria, said methodcomprising administering to said patient in need thereof a compoundwhich inhibits complement or inhibits formation of C5b-9.
 2. The methodof claim 1 wherein said quality of life is measured by a FACIT-Fatiguescore.
 3. The method, of claim 2 wherein the FACIT-Fatigue scoreincreases by at least 3 points.
 4. The method of claim 2 wherein theFACIT-Fatigue score increases by ≧4 points.
 5. The method of claim 1wherein said quality of life is measured by an EORTC QLQ-C30 score. 6.The method of claim 5 wherein said EORTC QLQ-C30 score improves by ≧10%of the pretreatment score.
 7. The method of claim 5 wherein said aspectof the quality of life as measured by an EORTC QLQ-C30 score is selectedfrom the group consisting of a) global health status, b) physicalfunctioning, c) emotional functioning, d) cognitive functioning, e) rolefunctioning, f) social functioning, g) fatigue, h) pain, i) dyspnea, j)appetite loss, and k) insomnia.
 8. The method of claim 7 wherein saidaspect of quality of life is fatigue.
 9. The method of claim 1 whereinsaid compound is selected from the group consisting of CR1, LEX-CR1,MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, andK76 COOH.
 10. The method of claim 1 wherein said compound is a steroidthat suppresses complement.
 11. The method of claim 1 wherein saidcompound is selected from the group consisting of antibodies, activeantibody fragments, soluble complement inhibitory compounds, proteins,soluble complement inhibitors with a lipid tail, protein fragments,peptides, small organic compounds, RNA aptamers, L-RNA aptamers,spiegelmers, antisense compounds, serine protease inhibitors, doublestranded RNA, small interfering RNA, locked nucleic acid inhibitors, andpeptide nucleic acid inhibitors.
 12. The method of claim 11 wherein saidcompound is an antibody or an active antibody fragment.
 13. The methodof claim 12 wherein said antibody or active antibody fragment isselected from the group consisting of a) polyclonal antibodies, b)monoclonal antibodies, c) single chain antibodies, d) chimericantibodies, e) humanized antibodies, f) Fabs, g) F(ab′)s, h) F(ab′)₂S,i) Fvs, j) diabodies, and k) human antibodies.
 14. The method of claim12 wherein said antibody or active antibody fragment blocks C5 cleavage.15. The method of claim 12 wherein said antibody or active antibodyfragment inhibits the formation of C5b-9.
 16. The method of claim 12wherein said antibody is eculizumab.
 17. The method of claim 12 whereinsaid antibody or active antibody fragment is administered for at least 6months.
 18. The method of claim 1 wherein said patient has aplasticanemia or myelodysplastic syndrome.
 19. The method of claim 1 whereinsaid patient is anemic.
 20. The method of claim 19 wherein said patientremains anemic following treatment.
 21. The method of claim 19 whereinsaid patient has a hemoglobin level less than i) 14 g/dL if a man or ii)12 g/dL if a woman.
 22. The method of claim 19 wherein said patient hasa hemoglobin level less than i) 13 g/dL if a man or ii) 11 g/dL if awoman.
 23. The method of claim 19 wherein said patient has a hemoglobinlevel less than i) 12 g/dL if a man or ii) 10 g/dL if a woman.
 24. Themethod of claim 1 wherein said compound inhibits intravascularhemolysis.
 25. The method of claim 1 wherein said method results in agreater than 30% reduction in LDH in said patient. 26-43. (canceled) 44.The method of claim 24 wherein said patient is anemic and said patientremains anemic after said administration. 45-67. (canceled)
 68. A methodof prolonging the health-adjusted life expectancy of a patientcomprising administering to said patient in need thereof a compoundwhich inhibits complement or inhibits formation of C5b-9.
 69. The methodof claim 68 wherein said patient is anemic.
 70. The method of claim 69wherein said patient remains anemic following treatment.
 71. The methodof claim 68 wherein said patient has a hemoglobin level less than i) 14g/dL if a man or ii) 12 g/dL if a woman.
 72. The method of claim 68wherein said patient has a hemoglobin level less than i) 13 g/dL if aman or ii) 11 g/dL if a woman.
 73. The method of claim 68 wherein saidpatient has a hemoglobin level less than i) 12 g/dL if a man or ii) 10g/dL if a woman.
 74. The method of claim 68 wherein said patient suffersfrom paroxysmal nocturnal hemoglobinuria.
 75. The method of claim 68wherein said health-adjusted life expectancy is measured according to aunit selected from the group consisting of Years of potential life lost,Disability-free life expectancy, Health-adjusted life year, Qualityadjusted life year, Healthy years equivalents, Healthy days gained,Episode-free day, Q-TWiST, Health Utilities Index, or Years of healthylife.
 76. The method of claim 75 wherein the health-adjusted lifeexpectancy in a subject is prolonged by at least one day.
 77. The methodof claim 75 wherein the health-adjusted life expectancy in a subject isprolonged by at least week.
 78. The method of claim 75 wherein thehealth-adjusted life expectancy in a subject is prolonged by at leastone month.
 79. The method of claim 75 wherein the health-adjusted lifeexpectancy in a subject is prolonged by at least one year.
 80. Themethod of claim 68 wherein said compound is selected from the groupconsisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venomfactor, FUT-175, complestatin, and K76 COOH.
 81. The method of claim 68wherein said compound is a steroid that suppresses complement.
 82. Themethod of claim 68 wherein said compound is selected from the groupconsisting of antibodies, active antibody fragments, soluble complementinhibitory compounds, proteins, soluble complement inhibitors with alipid tail, protein fragments, peptides, small organic compounds, RNAaptamers, L-RNA aptamers, spiegelmers, antisense compounds, serineprotease inhibitors, double stranded RNA, small interfering RNA, lockednucleic acid inhibitors, and peptide nucleic acid inhibitors.
 83. Themethod of claim 82 wherein said compound is an antibody or an activeantibody fragment.
 84. The method of claim 83 wherein said antibody oractive antibody fragment is selected from the group consisting of a)polyclonal antibodies, b) monoclonal antibodies, c) single chainantibodies, d) chimeric antibodies, e) humanized antibodies, f) Fabs, g)F(ab′)s, h) F(ab′)₂S, i) Fvs, j) diabodies, and k) human antibodies. 85.The method of claim 83 wherein said antibody or active antibody fragmentblocks C5 cleavage.
 86. The method of claim 83 wherein said antibody oractive antibody fragment is administered for at least 6 months.
 87. Themethod of claim 68 wherein said compound inhibits complement or inhibitsthe formation of C5b-9.
 88. The method of claim 87 wherein said compoundis an antibody or an active antibody fragment.
 89. The method of claim88 wherein said antibody is eculizumab.
 90. The method of claim 68wherein said patient has aplastic anemia or myelodysplastic syndrome.91. A method to improve at least one aspect of the quality of life of apatient suffering from paroxysmal nocturnal hemoglobinuria, said methodcomprising administering to said patient in need thereof a compoundwhich inhibits intravascular hemolysis.
 92. A method to improve at leastone aspect of the quality of life of an anemic patient whose anemiaresults at least in part from hemolysis, said method comprisingadministering to said patient in need thereof a compound which inhibitsintravascular hemolysis, wherein said patient remains anemic.